The well-understood catalytic cycle between horseradish peroxidase (HRP) and its commonly utilized colorimetric substrate, tetramethylbenzidine (TMB), proceeds as follows. First, HRP undergoes a two-electron oxidation of its ferriheme prosthetic group by hydrogen peroxide in solution. The oxidized HRP, known as compound-I, consists of an oxyferryl iron and a porphyrin τ cation radical [Everse, J., et al., Peroxidases in Chemistry and Biology. Vol. 1, 1991, Boca Raton: CFC Press. 620]. In the next reaction, the oxidized HRP converts the TMB in bulk solution from its normal reduced state to a colored oxidized form. Specifically, in a first step, a one-electron oxidation of TMB results in the formation of a cation radical intermediate. In a second reaction, the cation radical is further oxidized to form a yellow colored diimine. Two of the intermediate cation radicals may also combine to form a blue charge-transfer complex, which may be readily quantified with spectrophotometry [Josephy, P. D., et at. Journal of Biological Chemistry, 1982. 257(7): p. 3669-3675; Bally, R. W. and T. C. J. Gribnau, J Clin Chem Clin Biochem, 1989. 27(10): p. 791-796]. In the presence of dextran sulfate or other precipitating agents, the normally soluble oxidized form of TMB reacts to form an insoluble dark blue colored precipitate [McKimm-Breschkin, J. L., Journal of Immunological Methods, 1990. 135: p. 277-280]. As a result of the TMB oxidation steps, the enzyme is reduced to its native resting state, and may then be re-oxidized by hydrogen peroxide to restart the cycle. This peroxidase reaction sequence is well known in the literature and has been used for a number of detection schemes [Volpe, G., et al., Analyst, 1998. 123: p. 1303-1307; Alfonta, L., et at., Analytical Chemistry, 2001. 73(21): p. 5287-5295; Loo, R. W., et al., Analytical Biochemistry, 2005. 337(2): p. 338-342].
Direct electron transfer (DET) between an electrode material and redox-active biomolecules was first reported in 1977, and involved the use of cyclic voltammetry measurements of cytochrome-c etectrochemistry [Eddowes, M. J. and H. A. G. Hill, Journal of the Chemical Society—Chemical Communications, 1977. 21: p. 771-772; Yeh, P. and T. Kuwana, Chemistry Letters, 1977. 10: p. 1145-48]. Since these initial reports, efficient direct electron transfer has been documented for a number of redox enzymes, the majority of which contain a metallocenter, namely a heme group [Habermuller, L., et al. Fresenius Journal of Analytical Chemistry, 2000. 366(6-7): p. 560-568; Gorton, L., et al., Analytica Chimica Acta, 1999. 400: p. 91-108]. DET has also been established between HRP and a number of electrode surfaces, including carbon and graphite materials, gold, and platinum [Ruzgas, T., et at., Journal of Electroanalytical Chemistry, 1995, 391: p. 41-49]. HRP is one of the most commonly studied redox enzymes for coupling electron transport directly to conductive surfaces [Ruzgas, T., et al., Journal of Electroanalytical Chemistry, 1995. 391: p. 41-49; Yaropolov, A. I., et al. Bioelectrochemistry and Bioenergetics, 1978. 5(1): p. 18-24; Ferapontova, E., Electroanalysis, 2004. 16: p. 1101-1112]. It is known that this coupling can be used to monitor redox reactions mediated by HRP, such as those involved in biosensor applications, by monitoring current flow to an electrode onto which the HRP is adsorbed [Ghindilis, A. L., et al. Electroanalysis, 1997. 9(9): p. 661-674.].
There remains a need, however, for improved and more flexible methods of amplifying redox reactions used in chemical sensors. The invention disclosed herein addresses these needs and others by providing a means of exploiting electron transfer to remote sites via electrically coupled conductive surfaces.